A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

Abstract

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

Identification of Malassezia furfur Secreted Aspartyl Protease 1 (MfSAP1) and Its Role in Extracellular Matrix Degradation

“…N/TERT-1 keratinocytes (Dickson et al, 2000) were grown to ∼60% confluency in keratinocyte serum free media (Life Technologies) on 10 cm tissue cultured treated dishes and decellularized using freshly made 20 mM ammonium hydroxide following the protocol of Hellewell et al (2017). Primary dermal fibroblasts were cultured using the previously reported macromolecular crowding model (Lareu et al, 2007) in DMEM (Nacalai Tesque) medium containing 18.78 mg/ml Ficoll 70 (Sigma Aldrich), 12.5 mg/ml Ficoll 400 (Sigma Aldrich), and 100 µM ascorbic acid (Sigma Aldrich).…”

Characterization of the Extracellular Matrix of Normal and Diseased Tissues Using Proteomics

“…One critical step in this proteomic approach was the development of a method to enrich ECM proteins from tissues and tumors. To do so, we and others devised decellularization methods that deplete intracellular proteins and permit the enrichment of ECM proteins 8,[11][12][13][14][15][16][17][18][19][20] . A second critical step of the approach was the identification and annotation of the ECM components, or 'matrisome', in large proteomic datasets.…”

Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination fold

“…The lysate was incubated for 30 min on ice and cleared by centrifugation. For isolation of ECM-bound Shh, the decellularized tissue culture dish was washed with PBS and deionized water at least 5 times and scraped with a cell scraper and 5X SDS sample buffer heated to 95 o C, as described (Hellewell et al, 2017). A fifth of the sample was run on a 12% SDS-PAGE gel and transferred to a 0.45µ nitrocellulose membrane.…”